Rescuing the traffic-deficient mutants of rat μ-opioid receptors with hydrophobic ligands

Deletion of a sequence near the fifth transmembrane domain (²⁵⁸RLSKV²⁶², i3-1 mutant) and a motif residing at the proximal carboxyl tail (³⁴⁴KFCTR³⁴⁸, C-2 mutant) resulted in μ-opioid receptor mutants that were poorly expressed on the surface of transfected human embryonic kidney 293 cells. Treatment with the opioid antagonist naloxone, the agonist etorphine, and other hydrophobic ligands enhanced cell surface expression of i3-1 and C-2 mutants. The observed enhancement was time-and concentration-dependent, required the ligands to be membrane permeable, and was not the result of the reversal of the constitutive activities of the mutant receptors. The binding of the ligands resulted in the trafficking of the mutant receptors retained in the endoplasmic reticulum to the cell surface. The cell surface-expressed mutant C-2, but not i3-1, fully retained ability to mediate inhibition of adenylyl cyclase activity. Furthermore, the Golgi-disturbing agents brefeldin A and monensin completely blocked naloxone-enhanced expression of i3-1 and C-2 mutants. Results of these studies suggest that intracellular interactions of agonist and antagonist with mutant receptors can serve as chaperones in the trafficking of the mutants to the cell surface.